Snake Venomics of Central American Pitvipers: Clues for

Rationalizing the Distinct Envenomation Profiles of Atropoides

nummifer and Atropoides picadoi
Yamileth Angulo,† José Escolano,‡ Bruno Lomonte,† José María Gutiérrez,† Libia Sanz,‡ and
Juan J. Calvete*,‡

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica, and
Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11,
46010 Valencia, Spain

Received September 20, 2007; Accepted October 29, 2007

We report the proteomic characterization of the Central American pitvipers Atropoides nummifer and
Atropoides picadoi. The crude venoms were fractionated by reverse-phase high-performance liquid
chromatography (HPLC), followed by analysis of each chromatographic fraction by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, matrix-assisted laser
desorption ionization-time of flight (MALDI-TOF) mass fingerprinting, and collision-induced
dissociation-tandem mass spectrometry (CID-MS/MS) of tryptic peptides. Each venom contained a
number of bradykinin-potentiating peptides and around 25–27 proteins of molecular masses in the
range of 7–112 kDa, belonging to only nine different toxin families (disintegrin, DC fragment, snake
venom vascular endothelial growth factor, phospholipases A2, serine protease, cysteine-rich secretory
proteins, C-type lectins, L-amino acid oxidase, and Zn2+-dependent metalloproteases), albeit distinctly
distributed among the two Atropoides species. In addition, A. nummifer expresses low amounts of a
three-finger toxin not detected in the venom of A. picadoi. The major toxins of A. nummifer belong to
the PLA2 (relative abundance, 36.5%) and the serine proteinase (22%) families, whereas the most
abundant A. picadoi toxins are Zn2+-dependent metalloproteinases (66.4%). We estimate that the
similarity of venom proteins between the two Atropoides taxa may be around 14–16%. The high degree
of differentiation in the venom proteome among congeneric taxa emphasizes unique aspects of venom
composition of related species of Atropoides snakes and points to a strong role for adaptive
diversification via natural selection as a cause of this distinctiveness. On the other hand, their distinct
venom toxin compositions provide clues for rationalizing the low hemorrhagic, coagulant, and
defibrinating activities and the high myotoxic and proteolytic effects evoked by A. nummifer snakebite
in comparison to other crotaline snake venoms and the high hemorrhagic activity of A. picadoi.

Keywords: Atropoides nummifer • Atropoides mexicanus • Atropoides picadoi • Bitis caudalis • jumping
pitviper • Picadoi’s pitviper • snake venom protein families • proteomics • viperid toxins • snake venomics
• N-terminal sequencing • mass spectrometry • three-finger toxin

Introduction ticularly at the intergeneric level.1 Within Neotropical pitvipers,

Pitvipers (Crotalinae) are the most widely distributed sub-
family of the venomous snake family Viperidae, with major
radiations of its over 190 species (∼75% of viperid species
allocated in 29 genera) (http://www.reptile-database.org) in
both the Old and New Worlds.1 This diverse radiation has
received substantial taxonomic and phylogenetic attention over
the last several decades, yet the classification of the lineages
within the subfamily Crotalinae remains controversial,2,3 par-
* To whom correspondence should be addressed: Instituto de Biome-
dicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC),
Jaime Roig 11, 46010 Valencia, Spain. Telephone: +34-96-339-1778. Fax: +34-
96-369-0800. E-mail: jcalvete@ibv.csic.es.
†
Universidad de Costa Rica.
‡
Consejo Superior de Investigaciones Científicas (CSIC).
the Middle American lineage comprise genera Atropoides,
Cerrophidion, and Porthidium, which form a clade inferred to
be the sister group to a clade comprising the South American
genera Bothrocophias, Bothrops, and Bothriopsis.2,3
Atropoides is a genus of thick-bodied (greatest recorded
length of 120 cm) pit vipers feeding on small creatures, such
as frogs, rodents, and lizards. Of the three currently recognized
species of Atropoides, Atropoides olmec (Tuxtlan jumping
pitviper) occupies the smallest range, occurring only in the
Mexican Sierra de las Tuxtlas.1,4 The range of Atropoides picadoi
(Picadoi’s jumping pitviper) is more extensive. It is found in
the mountains of Costa Rica and western Panama at 50–1500
m altitude, including the Cordillera de Tilarán, the Cordillera
Central, and the Cordillera de Talamanca.1 Atropoides num-

708 Journal of Proteome Research 2008, 7, 708–719 10.1021/pr700610z CCC: $40.75  2008 American Chemical Society
Published on Web 12/28/2007
Proteomics of Atropoides Snake Venoms
mifer (Mexican jumping pitviper). inhabits the largest range,
from San Luis Potosí in eastern Mexico to central Panama and
can be found in various types of forest, including cloud forest
and rain forest, at 40–1600 m altitude. The common names
alludes to the ability that these snakes have to launch them-
selves at an attacker during a strike. Unlike most vipers,
members of this genus will strike and then hold on and chew.
However, the effects of the venom include only transient pain
and mild swelling. A. nummifer possesses one of the least toxic
pitviper venoms tested thus far in Central and South America.5
A. nummifer venom shows relatively low hemorrhagic, coagu-
lant, and defibrinating activities6 but displays higher myotoxic
and proteolytic effects than other crotaline snake venoms.7,8
In contrast, of 10 different Costa Rican pit viper venoms tested
on mice, that of A. picadoi was the most hemorrhagic.9,10
The distinct signs and symptoms of envenomation by A.
nummifer and A. picadoi snakebites suggests that venoms from
these species may contain different toxin repertoires. Venoms
represent the critical innovation in ophidian evolution that
allowed advanced snakes to transition from a mechanical
(constriction) to a chemical (venom) means of subduing and
digesting prey larger than themselves, and as such, venom
proteins have multiple functions, including immobilizing,
paralyzing, killing, and digesting prey. Venom toxins likely
evolved from proteins with normal physiological functions and
appear to have been recruited into the venom proteome before
the diversification of the advanced snakes, at the base of the
Colubroid radiation.11–14 Given the central role that diet has
played in the adaptive radiation of snakes,15 venom thus
represents a key adaptation that has played an important
function in the diversification of these animals. On the other
hand, venom composition may retain information on its
evolutionary history and may thus have a potential taxonomical
value.16 However, despite the fact that characterization of the
protein content of snake venoms may have a number of
potential benefits for basic research, clinical diagnosis, devel-
opment of new research tools and drugs of potential clinical
use and for antivenom production strategies,17 only a single
venom toxin sequence from any Atropoides species (PLA2
myotoxin II of A. nummifer, P82950)18 is annotated in the
SwissProt/TrEMBL nonredundant database (Release 37 of July
2007).
To address the need for detailed proteomic studies of snake
venoms, we have initiated a snake venomics project whose
long-term goal is the in-depth analysis of viperid venom
proteomes. To date, we have reported the protein composition
of the venoms from the North American rattlesnakes Sistrurus
miliarius barbouri19,20 and Sitrurus catenatus (subspecies cat-
enatus, tergeminus, and edwasdsii),20 the Tunisian vipers
Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina,21
and the Afrotropical species Bitis arietans (Ghana),22 Bitis
gabonica gabonica,23 Bitis gabonica rhinoceros, Bitis nasicornis,
and Bitis caudalis.16 Here, we report the proteomic character-
ization of the venoms of A. nummifer and A. picadoi.
Experimental Section
Isolation of Venom Proteins. Crude venoms of A. nummifer
(also denominated A. mexicanus; common name, jumping
viper) and A. picadoi (Picadoi’s pitviper) were pooled from at
least 20 specimens collected in Costa Rica and kept at the
serpentarium of the Instituto Clodomiro Picado, University of
Costa Rica in San José. For reverse-phase high-performance
liquid chromatography (HPLC) separations, 2–5 mg of crude,
research articles
lyophillized venom were dissolved in 100 µL of 0.05% trifluo-
roacetic acid (TFA) and 5% acetonitrile and insoluble material
was removed by centrifugation in an Eppendorff centrifuge at
13000g for 10 min at room temperature. Proteins in the soluble
material were separated using an ETTAN LC HPLC system
(Amersham Biosciences) and a Lichrosphere RP100 C18 column
(250 × 4 mm, 5 µm particle size) eluted at 1 mL/min with a
linear gradient of 0.1% TFA in water (solution A) and acetoni-
trile (solution B) (5% B for 10 min, followed by 5–15% B over
20 min, 15–45% B over 120 min, and 45–70% B over 20 min).
Protein detection was at 215 nm, and peaks were collected
manually and dried in a Speed-Vac (Savant). The relative
abundances (percent of the total venom proteins) of the
different protein families in the venoms were estimated from
the relation of the sum of the areas of the reverse-phase
chromatographic peaks containing proteins from the same
family to the total area of venom protein peaks.
Characterization of HPLC-Isolated Proteins. Isolated pro-
tein fractions were subjected to N-terminal sequence analysis
(using a Procise instrument, Applied Biosystems, Foster City,
CA), following the instructions of the manufacturer. Amino acid
sequence similarity searches were performed against the avail-
able databanks using the BLAST program24 implemented in the
WU-BLAST2 search engine at http://www.bork.embl-heidel-
berg.de. The molecular masses of the purified proteins were
determined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE; on 12–15% polyacrylamide gels)
and by electrospray ionization (ESI) mass spectrometry using
an Applied Biosystems QTrap 2000 mass spectrometer25 oper-
ated in enhanced multiple charge mode in the range of m/z
600–1700.
In-Gel Enzymatic Digestion and Mass Fingerprinting. Pro-
tein bands of interest were excised from a Coomassie Brilliant
Blue-stained SDS-PAGE and subjected to automated reduction
with dithiothreitol (DTT) and alkylation with iodoacetamide
and in-gel digestion with sequencing-grade bovine pancreas
trypsin (Roche) using a ProGest digestor (Genomic Solutions),
following the instructions of the manufacturer. A total of 0.65
µL of the tryptic peptide mixtures (total volume of ∼20 µL) was
spotted onto a matrix-assisted laser desorption ionization-time
of flight (MALDI-TOF) sample holder, mixed with an equal
volume of a saturated solution of R-cyano-4-hydroxycinnamic
acid (Sigma) in 50% acetonitrile containing 0.1% TFA, dried,
and analyzed with an Applied Biosystems Voyager-DE Pro
MALDI-TOF mass spectrometer, operated in delayed extrac-
tion and reflector modes. A tryptic peptide mixture of Cratylia
floribunda seed lectin (SwissProt accession code P81517)
prepared and previously characterized in our laboratory was
used as the mass calibration standard (mass range of 450–3300
Da).
Collision-Induced Dissociation-Tandem Mass Spectrom-
etry (CID-MS/MS). For peptide sequencing, the protein digest
mixture was loaded in a nanospray capillary column and
subjected to ESI mass spectrometric analysis using a QTrap
mass spectrometer (Applied Biosystems)25 equipped with a
nanospray source (Protana, Denmark). Doubly or triply charged
ions of selected peptides from the MALDI-TOF mass finger-
print spectra were analyzed in enhanced resolution MS mode,
and the monoisotopic ions were fragmented using the en-
hanced product ion tool with Q0 trapping. Enhanced resolution
was performed at 250 amu/s across the entire mass range. Settings
for MS/MS experiments were as follows: Q1, unit resolution;
Q1-Q2 collision energy, 30–40 eV; Q3 entry barrier, 8 V; linear
Journal of Proteome Research • Vol. 7, No. 2, 2008 709
research articles
ion trap (LIT) Q3 fill time, 250 ms; and Q3 scan rate, 1000 amu/s.
CID spectra were interpreted manually or using a licensed version
of the MASCOT program (http://www.matrixscience.com) against
a private database containing 927 viperid protein sequences
deposited in the SwissProt/TrEMBL database (Knowledgebase
Release 12 of July 2007; http://us.expasy.org/sprot/; 212 in
SwissProt, 715 in TrEMBL) plus the previously assigned peptide
ion sequences from snake venomics projects carried out in our
laboratory.16,19–23 MS/MS mass tolerance was set to (0.6 Da.
Carbamidomethyl cysteine and oxidation of methione were
fixed and variable modifications, respectively.
Variation in Venom Composition between Atropoides
Taxa. We used similarity coefficients to estimate the similarity
of venom proteins between taxa. These coefficients are similar
to the bandsharing coefficients used to compare individual
genetic profiles based on multilocus DNA fingerprints.26 We
defined the protein similarity coefficient (PSC) between two
species “a” and “b” in the following way: PSCab ) [2 × (number
of proteins shared between a and b)/(total number of distinct
proteins in a + total number of distinct proteins in b)] × 100.
We judged two proteins (listed in Tables 1-3) as being different
when they met one or more of these criteria: (1) had different
N-terminal sequences and/or distinct internal peptides se-
quences (derived from MS/MS data) corresponding to homolo-
gous regions, (2) had different peptide mass fingerprints, (3)
were of different sizes [judged by MALDI-TOF MS or SDS-
PAGE; For these comparisons, two proteins were judged to
differ in size if they differed by more than our estimate of the
95% confidence interval for particular sizing techniques (0.01%
for ESI-QTrap MS, 0.4% for MALDI-TOF MS-derived masses,
and 1.4 kDa for SDS-PAGE-determined masses)], or (4) eluted
in different reverse-phase HPLC peaks. We emphasize that
these measures will give only minimum estimates of the
similarities between the venom profiles. We suspect that a
number of the proteins that we judge to be the same using the
above criteria would be found to differ at one or more of these
criteria if more complete information were available.
Results and Discussion
Characterization of the Venom Proteomes of A. nummi-
fer and A. picadoi. The crude venoms of A. nummifer and A.
picadoi were fractionated by reverse-phase HPLC (Figures 1
and 3), followed by analysis of each chromatographic fraction
by SDS-PAGE (Figures 2 and 4), N-terminal sequencing, and
MALDI-TOF mass spectrometry (Tables 1 and 2). Protein
fractions showing single electrophoretic band, molecular
mass, and N-terminal sequence were straightforwardly as-
signed by BLAST analysis (http://www.ncbi.nlm.nih.gov/
BLAST) to a known protein family, indicating that, although
a single toxin from A. nummifer (myotoxin II, P82950) is
annotated in the public-accessible databases, representative
members of most snake venom toxin families are present
among the 927 viperid protein sequences deposited to date
in the SwissProt/TrEMBL database (Knowledgebase Release
12 of July 2007; http://us.expasy.org/sprot/). Protein fractions
showing heterogeneous or blocked N termini were analyzed
by SDS-PAGE, and the bands of interest were subjected to
automated reduction, carbamidomethylation, and in-gel
tryptic digestion in a ProGest digestor (Genomic Solutions).
The resulting tryptic peptides were then analyzed by
MALDI-TOF mass fingerprinting followed by amino acid
sequence determination of selected doubly and triply charged
peptide ions by CID-MS/MS. Except for the protein eluting
710 Journal of Proteome Research • Vol. 7, No. 2, 2008
Angulo et al.
in peak 22, which was identified as a PLA2 molecule highly
related to myotoxin II,18 the peptide mass fingerprinting
approach alone was unable to identify any protein in the
databases. In addition, as expected from the rapid amino
acid sequence divergence of venom proteins evolving under
accelerated evolution,27–32 with a few exceptions, the product
ion spectra did not match any known protein using the
ProteinProspector (http://prospector.ucsf.edu) or the MAS-
COT (http://www.matrixscience.com) search programs. Fur-
thermore, it was not too unusual that a product ion spectrum
matched with high MASCOT score to a particular peptide
sequence corresponds actually to a tryptic peptide of an
homologue snake toxin containing one or more nearly
isobaric amino acid substitutions. Hence, it is necessary to
revise manually all of the CID-MS/MS spectra (to confirm
the assigned peptide sequence or for performing de novo
sequencing) and submit the deduced peptide ion sequences
to BLAST similarity searches. Although the lack of any
complete snake genome sequence is a serious drawback for
the identification of venom proteins, high-quality MS/MS
peptide ion fragmentation spectra usually yield sufficient
amino acid sequence information derived from almost the
complete series of sequence-specific b and/or y ions to
unambiguously identify a homologue protein in the current
databases. The outlined snake venomics approach allowed
us to assign unambiguously all of the isolated venom toxins
representing over 0.05% of the total venom proteins to
known protein families (Tables 1 and 2).
Supporting the view that venom proteomes are mainly com-
posed of proteins belonging to a few protein families,12–14,16,19–23
the proteins found in the venoms of A. nummifer and A. picadoi
cluster, respectively, into 11 and 10 different toxin families
(bradykinin-potentiating peptides, disintegrin, DC fragment,
snake venom vascular endothelial growth factor (svVEGF),
PLA2, serine protease, cysteine-rich secretory proteins (CRISP),
C-type lectins, L-amino acid oxidase (LAO), and Zn2+-depend-
ent metalloproteases), albeit each species exhibiting distinct
relative abundances, which are listed in Table 3.
Large Venom Toxin Composition Variation between Atro-
poides Species: Occurrence of a Three-Finger Toxin in A.
nummifer Venom. As judged from the data shown in Tables 1
and 2, the venom proteomes of A. nummifer and A. picadoi
comprise a similar number, around 25–27, of distinct proteins
(Figure 5). However, whereas the major toxins of A. nummifer
belong to the PLA2 and the serine proteinase families, the most
abundant A. picadoi toxins are Zn2+-dependent metallopro-
teinases (Table 3). Among the major A. nummifer PLA2 mol-
ecules, the protein eluting in peaks 22 and 23 (Table 1) exhibits
similar chromatographic retention times, molecular mass, and
N-terminal sequences as the PLA2 molecules from A. picadoi
venom eluting in peaks 15 and 18 (Table 2). Similarly, the
galactose-specific lectin found in each venom (An-27–30, Table
1 and Ap-21, Table 2) share chromatographic behavior, N-
terminal sequence, molecular mass, and tryptic peptide ion
sequences, and the same is true for the LAO proteins isolated
in fractions An-35 (Figure 1 and Table 1) and Ap-27 (Figure 2
and Table 2) and the svVEGF (An-20 and Ap-14). On the other
hand, the major PLA2 molecule eluted in peak An-21 (Table
1), which may correspond to the myotoxin I described by Rojas
and co-workers,5 is uniquely expressed in A. nummifer venom.
Similarly, among the major proteins found in Sistrurus venoms,
PLA2 proteins appear to be exceptionally divergent at both the
intra- and interspecific level,20 suggesting that they have been
Proteomics of Atropoides Snake Venoms research articles
Table 1. Assignment of the Reverse-Phase Fractions of A. nummifer (A. mexicanus) Venom, Isolated as in Figure 1, to Protein
Families by N-Terminal Edman Sequencing, Mass Spectrometry, and Collision-Induced Fragmentation by nESI-MS/MS of Selected
Peptide Ions from In-Gel-Digested Protein Bands (Separated by SDS-PAGE as in Figure 2)a
HPLC fraction N-terminal molecular peptide ion MS/MS-derived protein
An- sequence mass m/z z sequence family
1–4, 6, np
8, 9, 13
5 ND 370.6 2 (177.7)APAAPH bradykinin-
potentiating
peptides
348.8 2 PHHIPP
7 ND 532.4 2 TPPAGPDVGPR bradykinin-
potentiating
peptide
10 ND 616.6 2 LGTPPAGPDVGPR bradykinin-
potentiating
peptide
488.6 2 ZRFPQYR Zn2+-metalloprotease
fragment
11 EAGEECDCGAPTNP 7710.3 684.3 3 LRPGAQCAEGL medium-size
CCDAAT CCDQCR disintegrin
508.2 2 ARGDNPDDR
PPPISPP 704.3 bradykinin-
potentiating
peptide
12 EECDCGAPTNPC 7452.3 medium-size
CDAATCKL disintegrin
ECDCGAPTNPCCD 7323.2 684.3 3 LRPGAQCAEGL
AATCKLR CCDQCR
508.2 2 ARGDNPDDR
14 ND 630.2 2 PPGPVGPDR(223.0) unknown
397.2 2 PPGPPIPP bradykinin-
potentiating
peptide
15 blocked 622.8 2 ZKWDPPPISPP bradykinin-
potentiating
peptide
16 LICEDCSLPNCD 6833* 712.3 three-finger toxin
FLPGIL
17 ND 24 062* 635.7 2 LYCFPSSPGDK DC fragment
712.3 3 (953.4)CDCGS
PATCR
18 ND 24 390* 635.7 2 LYCFPSSPGDK DC fragment
19 ND 14 409* 650.1 2 GXYGCNCGVGSR PLA2
20 ND 38 kDa9/36 kDa1 685.2 2 ZVMPFMEVYSR svVEGF
13 kDa1, 8 kDa1 647.3 2 XDATCVCVXSR
682.3 2 AXTMEGNQASWR
34 kDa9/16 kDa1 650.1 2 GLYGCNCGVGSR PLA2
640.4 3 NAIASYGLYGC
NCGVGSR
21 SLYELGKMILQET 13 751.0 650.1 2 GXYGCNCGVGSR PLA2 myotoxin I
GKNAIA
640.4 3 NAIASYGLYGC
NCGVGSR
22 NLYQLWKMIL 13 792.5 482.6 2 NLYQLWK PLA2 myotoxin II
QETGKNA [P82950]
569.1 2 TDSYSYSWK
925.4 2 NAAPSYGFY
GCNCGVGR
509.9 2 IYPKPLCK
702.4 2 TIVCGKNNPCLK
751.8 2 CCYKALTDCSPK
460.2 2 MILQETGK
491.3 2 DNLDTYNK
23 SLIQFETLIMKIAGR 13 728* 479.6 2 YWFFPAK PLA2
669.9 2 SLIQFETLIM(ox)K
874.8 2 QXCECDKDAAXCXR

Journal of Proteome Research • Vol. 7, No. 2, 2008 711

research articles Angulo et al.
Table 1. Continued
HPLC fraction N-terminal molecular peptide ion MS/MS-derived protein
An- sequence mass m/z z sequence family
24
M SVDFDSESPRKPEIQ 24 744* CRISP
m VIGGDECNINEHRFL 38 kDa9/1 serine proteinase
25 SLIQFETLIMKIAGQ 13 740* PLA2
VIGGDECNINEHRFL 28 kDa9/(14 + 17 kDa)1 two-chain
KNxGLDKDIMLIRLR serine proteinase
26
M VIGGDECNINEHRFL 30–33 kDa9/1 678.9 2 (203.2)XVLTAAHCNR serine proteinase
812.9 2 CANINILDYIVCR
650.8 2 GGDECNINEHR
611.2 2 FLVALYDSHR
508.6 2 (157.7)FPTXPER
619.2 2 XNXNGYEVCR
m SLIQFETLIMKIAGQ 13 740* PLA2
27
M V(I/F)GGDECNINEHR(S/F)L 26 kDa9/1 serine proteinase
m NNCPQDWLPMNGLCY 28 kDa9/13 kDa1 621.3 2 DFSWEWTDR Gal-specific lectin
28
m VIGGDECNINEHRSL 66 kDa9/33 kDa1 679.3 2 (316.2)VLTAAHCNR serine proteinase
756.6 2 VIGGDECNINEHR
27–30 NNCPQDWLPMNGLCY 28 kDa /13 kDa
9 1
621.6 2 DFSWEWTDR Gal-specific lectin
784.3 2 EFCVEXVSHAGYR
29 VIGGDECNINEHRSL 28 kDa9/1 648.2 2 XNXXDYQVCR serine proteinase
757.6 2 VIGGDECNINEHR
539.3 2 (145.1)YAGXPVCR
736.6 3 PVEXTPVSXPSNPPXGSVCR
30 V(V/I)GGDECNINEHRSL 28–34 kDa9/1 764.3 2 IIGGDECNINEHR serine proteinase
704.3 2 FXVAXHDYWSR
877.8 2 TXCAGVXEGSTDTCDR
31 IIGGDECNINEHRFL 34 kDa9/1 640.6 2 XDXFDNEVCR serine proteinase
868.8 2 XXCAGVXEGGTDTCNR
717.6 3 (1003.3)PFQGXVSWGR
32 IIGGDECNINEHRFL 29 kDa9/1 640.6 2 XDXFDNEVCR serine proteinase
868.8 2 XXCAGVXEGGTDTCNR
717.6 3 (1003.3)PFQGXVSWGR
33–41 blocked 26 kDa9/1 783.8 3 XSHHDAQXXTAXVFDQNTXGR PI metalloprotease
676.8 3 SMNXHXSXNDVEXWSNR
34–41 ND 112 + 56 kDa /56 kDa
9 1
532.2 2 NPXEECFR L-amino acid
oxidase
758.3 2 ETDYEEFXEXAR
641.3 2 SAGQXYQESXGK
634.8 2 VGEVNQDPGXXK
684.0 3 DCGDXVXNDXSXXHQXPK
37–40 ND 14–16 kDa1 783.8 3 XSHHDAQXXTAXVFDQNTXGR PI metalloprotease
fragment
676.8 3 SMNXHXSXNDVEXWSNR
40 ND 110 kDa9/42 kDa1 900.3 2 (200.2)GQCAEGXCCEQCK PIII metalloprotease
636.1 2 XYCFWSPGSR
502.4 2 (289.2)PFQPVK
41 ND 78 kDa9/42 kDa1 900.3 2 (200.2)GQCAEGXCCEQCK PIII metalloprotease
636.1 2 XYCFWSPGSR
a
X, Ile or Leu; Z, pyrrolidone carboxylic acid; M(ox), methionine sulfoxide. Unless otherwise stated, for MS/MS analyses, cysteine residues were
carbamidomethylated. Molecular masses of native proteins were determined by ESI ((0.02%) or MALDI-TOF (/) ((0.2%) mass spectrometry. Apparent
molecular mass determined by SDS-PAGE of nonreduced (9) and reduced (1) samples. np ) nonpeptidic material found. M and m, denote mayor and
minor products within the same HPLC fraction. The only previously reported venom protein from any Atropoides species is identified by its databank
accession code.

the subject of strong balancing selection33 within and diversify-
ing selection between taxa. Other studies have also shown that
PLA2 genes show high levels of divergence between species and
high levels of protein diversity at the population level.28,34–40
Serine proteinases and metalloproteinases have also di-
verged to a point where no structural similarity can be
discerned between these toxins of A. nummifer and A.
picadoi. In addition, A. nummifer venom departs from that
of A. picadoi by the distinct occurrence in the former of a
three-finger toxin (3FTx) (reverse-phase HPLC fraction An16
in Figures 1 and 2 and Table 1). Three-finger toxins appear
to be widely distributed in Elapidae (including Hydrophiinae)
venoms41 and typically block nicotinic acetylcholine recep-
tors, resulting in postsynapyic neurotoxicity.42 Recently,
three-finger toxins have also been described in Viperidae
(Daboia russelli43 and Lachesis muta44) as well as Colubridae
(Boiga dendrophila).45,46 These observations, along with our
finding of a 3FTx in A. nummifer venom, indicate that this
toxin family may be more widely distributed than previously
thought and supports the proposal that venom toxins likely
evolved from proteins with normal physiological functions
recruited into the venom proteome before the diversification
of the advanced snakes, at the base of the Colubroid
radiation.11–14 The N-terminal sequence, molecular mass,
and pharmacological activities of the Daboia russelli venom
3FTx indicate that it is a short-chain neurotoxin such as that
found in Elapid venom.43 On the other hand, the colubrid
3FTx (termed denmotoxin) represents a bird-specific neu-

712 Journal of Proteome Research • Vol. 7, No. 2, 2008

Proteomics of Atropoides Snake Venoms research articles
Table 2. Assignment of the Reverse-Phase Fractions of A. picadoi Venom, Isolated as in Figure 3, to Protein Families by
N-Terminal Edman Sequencing, Mass Spectrometry, and Collision-Induced Fragmentation by nESI-MS/MS of Selected Peptide
Ions from In-Gel-Digested Protein Bands (Separated by SDS-PAGE as in Figure 4)a
HPLC fraction peptide ion
An- N-terminal sequence molecular mass m/z z MS/MS-derived sequence protein family
1, 2, 6 np
3 ND 589.9 2 DTPPAGPDVGR bradykinin-potentiating peptide
4
m ND 489.9 2 ZQYNPYR unknown
5
m ND 395.9 2 FXQNXR unknown
7 ND 634.9 2 VGEVNQDPGXXK LAO fragment
8 ND 625.9 2 (215.1)WPPGHHIPP bradykinin-potentiating peptide
9 ND 537.9 2 (321.3)PGHHIPP bradykinin-potentiating peptide
10 ND 555.3 2 XTAPFESS(277.1) unknown
11 ECDCGAPTNPCCDAAT 7341.2 disintegrin
12 ND 23.5 kDa9/1 (DC fragment)
13 ND 23.5 kDa9/1 (DC fragment)
14 ND 26 kDa9/13 kDa1 685.2 2 ZVMPFMEVYSR svVEGF
18 kDa9, 10 kDa1 647.3 2 XDATCVCVXSR
682.3 2 AXTMEGNQASWR
556.2 2 NPNPVPTGCR
15 SLYQLWKMILQETGK 13 897.6 650.2 2 GLYGCNCGVGSR PLA2
469.2 2 SXYQXWK
568.7 2 TDSYSYSWK
16 SLIQFETLIMKIAGR 13 806.6 884.8 2 TXCECDKDAAXCFR PLA2
479.6 2 YWFFPAK
669.9 2 SLIQFETLIM(ox)K
17 NVDFDSESPRKPEIQ 24 787.1 583.2 2 NVDFDSESPR CRISP
769.3 2 MEWYPEAAANAER
635.9 3 KPEIQNEIVDLHNSLR
18 SLIQFETLIMKIAGR 13 790.6 479.6 2 YWFFPAK PLA2
669.9 2 SLIQFETLIM(ox)K
884.8 2 TXCECDKDAAXCFR
19 VVGGDECNINEHRSL 28 kDa9/1 serine proteinase
20 VVGGDECNINEHRSL 39.2 Da9/1 616.3 2 VKLNEDEQTR serine proteinase
749.9 2 VVGGDECNINEHR
467.8 2 XXCHXNR
523.9 2 VXYQDAXPK
21 VFGGDECNINEHRSL 29 kDa9/1 605.8 2 INILDHAVCR serine proteinase
773.8 2 VFGGDECNINEHR
552.2 2 VLNEDEQTR
NNCPQDWLPMNGLCY 26 kDa9/13 kDa1 621.6 2 DFSWEWTDR Gal-specific lectin
783.9 2 EFCVEXVSHAGYR
573.2 2 NQPDHYQNK
639.9 2 LWNDQVCESK
670.8 3 NNCPQDWLPMNGLCYK
22 VFGGDECNINEHRSLVVLF 27.5 kDa9/1 605.8 2 INILDHAVCR serine proteinase
773.8 2 VFGGDECNINEHR
552.2 2 VLNEDEQTR
19.5 kDa 605.8 2 INILDHAVCR serine proteinase
NNCPQDWLPMNGLCY 26 kDa9/13 kDa1 621.6 2 DFSWEWTDR Gal-specific lectin
784.1 2 EFCVEXVSHAGYR
23 VIGGDECNINEHRFL 30 kDa9/1 756.9 2 VIGGDECNINEHR serine proteinase
698.6 2 AAYPEYGXPATSR
610.9 2 FXVAXYDSHR
647.9 2 XNXXDYEVCR
24 VVGGDECNINEHRFL 28 kDa9/1 679.4 2 FXVAXYTFDXR serine proteinase
486.2 2 (212.2)PNQDER
728.4 2 SXPSSPPXVGSVCR
25–27 TPEHQRYIELVIVVD 22.9 kDa/ 555.8 2 TXDSFGEWR PI metalloproteinase
534.3 2 YNDGSDKXR
27 ANDRNPLEECFRETD 56 kDa9/1 532.2 2 NPXEECFR L-amino acid oxidase
758.3 2 ETDYEEFXEXAR
28 heterogeneous 58 kDa9/1 633.3 2 (145.2)VGPXQDHSPR PIII metalloproteinase
488.7 2 GSNYGYCR
110 kDa9/16 kDa1 503.8 2 TPFAAACXR C-type lectin-like
709.3 2 HWADAEWFCAR
29 heterogeneous 68 kDa9/1 670.4 2 YVEXVXVADYR PIII metalloproteinase

Journal of Proteome Research • Vol. 7, No. 2, 2008 713

research articles Angulo et al.
Table 2. Continued
HPLC fraction peptide ion
An- N-terminal sequence molecular mass m/z z MS/MS-derived sequence protein family
678.6 2 VAXVGXQXWSNR
46 kDa9/1 514.8 2 XPCAPEDVK PIII metalloproteinase
526.8 2 GNYYGYCR
801.6 2 MYEXANTVNDXYR
30 heterogeneous 28 kDa9 521.2 2 YYAWIGLR C-type lectin-like
453.7 2 TWEDAER
640.3 3 DCPSDWSSYEGHCYR
621.2 2 DCPSDESSYEGHCYR
16 kDa1 621.2 2 DCPSDESSYEGHCYR C-type lectin-like R subunit
14 kDa1 640.3 3 DCPSDWSSYEGHCYR C-type lectin-like β subunit
31 heterogeneous 42 kDa9/1 852.3 2 SDVDYTXNSFAEWR PIII metalloproteinase
626.8 3 (367.2)PDYCTGQSGDCPR
16 kDa1 521.2 2 YYAWIGLR C-type lectin-like
14 kDa1 851.8 2 SDVDYTXNSFAEWR metalloproteinase fragment
a
X, Ile or Leu; Z, pyrrolidone carboxylic acid; M(ox), methionine sulfoxide. Unless otherwise stated, for MS/MS analyses, cysteine residues were
carbamidomethylated. Molecular masses of native proteins were determined by ESI ((0.02%) or MALDI-TOF (/) ((0.2%) mass spectrometry. Apparent
molecular mass determined by SDS-PAGE of nonreduced (9) and reduced (1) samples. np, nonpeptidic material found. M and m, denote mayor and
minor products within the same HPLC fraction.

Table 3. Overview of the Relative Occurrence of Proteins (in

Percentage of the Total HPLC-Separated Proteins) of the
Different Families in the Venoms of A. nummifer
(A. mexicanus) and A. picadoi
percent of total venom proteins

protein family A. nummifer A. picadoi

bradykinin-potentiating peptides 8.6 1.8
medium-size disintegrin 2.5 <0.1
three-finger toxin <0.1 nda
DC fragment <0.1 <0.1
sv-VEGF <0.1 <0.1
PLA2 36.5 9.5
serine proteinase 22.0 13.5
CRISP 1.9 4.8
C-type lectin-like 1.3 1.8
L-amino acid oxidase 9.1 2.2
Zn2+-metalloproteinases
PI SVMP 14.8 9.7 Figure 1. Reverse-phase HPLC separation of the A. nummifer
PIII SVMP 3.4 56.7 venom proteins. A total of 2 mg of A. nummifer venom was
applied to a Lichrosphere RP100 C18 column, which was then
a
nd, not detected. developed with the following chromatographic conditions: iso-
cratically (5% B) for 10 min, followed by 5–15% B for 20 min,
rotoxin, which displayed potent postsynaptic neuromuscular 15–45% B for 120 min, and 45–70% B for 20 min. Fractions were
activity and irreversibly inhibited indirectly stimulated twitches collected manually and characterized by N-terminal sequencing,
in chick biventer cervicis nerve-muscle prepara- ESI mass spectrometry, tryptic peptide mass fingerprinting, and
tions.46,47 The biological activity of the 3FTx present in the CID-MS/MS of selected doubly or triply charged peptide ions.
The results are shown in Table 1.
venoms of the neotropical pitviper A. nummifer deserves
further investigation.
Using a similarity coefficient, we estimate that the similarity
of venom proteins between the two Atropoides taxa may be versial, particularly at the intergeneric level.3 In particular, the
around 14–16%. Furthermore, the large divergence between the genus Atropoides was inferred through Bayesian phylogenetic
two Atropoides species is highlighted by the fact that the methods to be paraphyletic with respect to Cerrophidion and
metalloproteinases of A. nummifer venom not only represent Porthidium, because of A. picadoi being distantly related to
around 27% of the amount of these toxins in the venom of A. other Atropoides species.2,3,48 Our proteomic study supports
picadoi but also belong to different structural classes (P-I in A. the large divergence among A. nummifer and A. picadoi. In a
nummifer versus P-III in A. picadoi) (Table 3). The high degree previous study of Bitis venoms,16 we showed that venom
of differentiation in the venom proteome among congeneric composition appears to keep information on the evolutionary
taxa emphasizes unique aspects of venom composition of history of congeneric taxa, which may be useful for resolving
related species of Atropoides snakes and points to a strong role or supporting cladogenetic events inferred fom DNA sequence
for adaptive diversification via natural selection as a cause of data. Therefore, to assess phylogenetic alliances between A.
this distinctiveness, whose possible physiological significance picadoi and the Cerrophidion and Porthidium clades requires
is discussed below. a detailed analysis of the venom proteomes of Middle American
Despite the efforts of numerous authors, phylogenetic species from these genera occupying geographically close
relationships within the subfamily Crotalinae remain contro- ecological niches, i.e., Porthidium nasutum, Porthidium ophry-

714 Journal of Proteome Research • Vol. 7, No. 2, 2008

Proteomics of Atropoides Snake Venoms
Figure 2. SDS-PAGE of reverse-phase separated fractions from the venom of A. nummifer. SDS-PAGE showing the protein composition
of the reverse-phase HPLC-separated venom protein fractions run under nonreduced (A) and reduced (B) conditions. Molecular mass
markers (in kilodaltons) are indicated at the left of each gel. Protein bands were excised and characterized by mass fingerprinting and
CID-MS/MS. The results are shown in Table 1.
Figure 3. Reverse-phase HPLC separation of the venom proteins
of A. picadoi. A total of 2 mg of A. picadoi venom were applied
to a Lichrosphere RP100 C18 column, which was then developed
as in Figure 1. Fractions were collected manually and character-
ized by N-terminal sequencing, ESI mass spectrometry, tryptic
peptide mass fingerprinting, and CID-MS/MS of selected doubly
or triply charged peptide ions. The results are shown in Table 2.
omegas, Porthidium volcanicum, and Cerrophidion godmani
(http://www.icp.ucr.ac.cr/serpient.htm). Among these species,
currently, only three venom proteins from Cerrophidion god-
mani are annotated in the protein databases, the N6 basic PLA2
research articles
Q6EER5 (N-terminal sequence, NLLQFNKMIKIMTKK) and the
K49 PLA2 molecules P81165 (N-terminal sequence, SMYQL-
WKMILQETGK) and Q8UVU7 (N-terminal sequence, SVYEL-
GKMILQETGK).
Clues for Rationalizing the Distinct Envenomation
Profiles of A. nummifer and A. picadoi. Although documenta-
tion of human accidents by A. nummifer and A. picadoi
snakebites is scarce, our proteomic study showing the drastic
divergences in the composition of the venoms from these two
species strongly suggests that the clinical manifestations of
envenomations by these two Atropoides species are likely to
differ significantly, with the venom of A. nummifer inducing
stronger myonecrosis and the venom of A. picadoi inducing
stronger hemorrhagic effects. Thus, the low hemorrhagic,
coagulant, and defibrinating activities6,10 and the high myotoxic
and proteolytic effects evoke in mice by A. nummifer venom
in comparison to other crotaline snake venoms7 can be
straightforwardly explained by the large PLA2 and serine
proteinase content of its venom. Myotoxic PLA2 molecules
account for most of the muscle necrosis that results from
envenenomation by crotaline snakes.49,50 The two major PLA2
molecules present in A. nummifer (An-21 and An-22, Figure 1
and Table 1) venom possess the signature residues of myotoxic
PLA2 molecules. Hence, the tryptic mass fingerprint of An-22
matches, with the exception of a S/R substitution at position
32, that of PLA2 myotoxin II [P82950].18 However, the molecular
mass of PLA2 An-22 (13 792.5 Da) departs from the isotope-
averaged molecular mass calculated for myotoxin II (13 698.7
Journal of Proteome Research • Vol. 7, No. 2, 2008 715
research articles Angulo et al.

Figure 4. SDS-PAGE of reverse-phase separated fractions from the venom of A. picadoi. SDS-PAGE showing the protein composition
of the reverse-phase HPLC-separated venom fractions (see Figure 3) run under nonreduced (A) and reduced (B) conditions. Molecular-
mass markers (in kilodaltons) are indicated at the left of each gel. Protein bands were excised and characterized by mass fingerprinting
and CID-MS/MS. The results are shown in Table 2.

Figure 5. Overall protein composition of Atropoides venoms. Comparison of the protein composition of the venoms of A. nummifer
(A) and A. picadoi (B). BPP, bradykinin-potentiating; DISI, disintegrin; PLA2, phospholipase A2; CRISP, cysteine-rich secretory protein;
C-lectin, C-type lectin-like protein; SVMP, snake venom metalloproteinase; LAO, L-amino acid oxidase; DC fragment, disintegrin/cysteine-
rich fragment; svVEGF, snake venom vascular endothelial growth factor.

Da, assuming that the 14 cysteine residues are engaged in
disulphide bonds), suggesting that An-22 and P82950 are closely
related. Altogether, the tryptic peptides sequenced by MS/MS
correspond to a sequence coverage of 67%, including the
conserved residues (L5, Q11, N28, R34, K49, K53, and T56) of
K49 PLA2 molecules from a large variety of pitvipers from the
genera Bothrops, Agkistrodon, Bothriechis, Crotalus, Trimeresu-
rus, and Protobothrops.49–52
The first 58 amino acid residues of the PLA2 molecule
eluted in peak 21 were determined by direct N-terminal

716 Journal of Proteome Research • Vol. 7, No. 2, 2008

Proteomics of Atropoides Snake Venoms
sequencing by Edman degradation of the reduced and
carboamidomethylated protein: SLYELGKMILQETGKNA-
IASYGLYGCNCGVGSRGKPKDATDRCCFMHKCCYKKLTACN.
This sequence is identical to the N-terminal sequence reported
for myotoxin I from A. nummifer,5 and its molecular mass
(13 751 Da) is close to the mass determined by ESI-MS (14 738
Da) for a K49 PLA2 from the same species by Tsai et al.53 Other
close homologues of An-21 are the K49 myotoxic PLA2 mol-
ecules I and II from Cerrophidion godmani [Q8UVU7]4,5 (87%
identity and 91% similarity), and similar to these molecules,
they exhibit the signature residues (underlined) of myotoxic
PLA2 molecules.
Our results stressing the possible biological relevance of
myotoxic PLA2 in the pathophysiology of A. nummifer enveno-
mation may find applications in the production and improve-
ment of antivenoms. We argue that a small set of antibodies
generated against the structural determinants identified in the
C-terminal loop region of the PLA2 scaffold and involved in
the determination of myotoxic and membrane-damaging
activities54,55 may neutralize the pharmacological effects of
myotoxins I and II. In line with this hypothesis, Calderón and
Lomonte55 have shown that although the C-terminal 115KKYRY-
YLKPLCKK129 region of myotoxin II of Bothrops asper venom
[P24605] does not represent an immunodominant B-cell epitope
in mice immunized against the purified toxin, animals chal-
lenged with the synthetic peptide 115–129 produced antibodies,
which cross-reacted with myotoxin II, and acquired a signifi-
cant protection against the development of myonecrosis.
The low hemorrhagic, coagulant, and defibrinating activities
of the A. nummifer venom may reflect its relatively low
amounts of Zn2+-dependent snake venom metalloproteinases
(SVMP) (Table 3). SVMP, also called hemorrhagins, display a
wide range of biological activities, including hemorrhagic,
fibrinolytic, activation of prothrombin, and factor X.56,57 SVMP
have been classified according to their domain structure.57–59
PI metalloproteinases (20–30 kDa) are single-domain proteins
with relatively weak hemorrhagic activity. The class PII met-
alloproteinases (30–60 kDa) contain a disintegrin domain at
the carboxyl terminus of a metalloproteinase domain structur-
ally similar to that in the PI class. Hemorrhagins of the PIII
class are large toxins (60–100 kDa) that comprise multidomain
enzymes built up by a N-terminal metalloproteinase domain
and C-terminal disintegrin-like and cysteine-rich domains. In
general terms, PIII SVMP are the more potent hemorrhagic
toxins60 and, in contrast to PI SVMP, which mainly produce
local hemorrhage, are capable of inducing systemic bleeding.
The distinct hemorrhagic potential of PIII versus PI SVMP,
along with the different amount and distribution of these toxins
among the venoms of A. nummifer (81% PI and 19% PIII of
total SVMP) and A. picadoi (15% PI and 85% PIII) (Table 3),
may explain the low hemorrhagic activity of the former and
the reported higher hemorrhagic effect of A. picadoi venom
among Costa Rican snake venoms.10
Concluding Remarks
A. nummifer and A. picadoi are so closely related in shape
and color pattern that it is difficult to distinguish one species
from the other on morphological grounds. However, variation
in the electrophoretic, biochemical, and functional properties
between their venoms have been noticed for more than 55
years.61,62 Our proteomic work represents a detailed analysis
of the venom composition of two Central American snakes
whose phylogenetic alliance still remains controversial. The
research articles
large degree of compositional variation between the venoms
of A. nummifer and A. picadoi supports the need of reassessing
the evolutionary relationships of these snakes. To this end, we
are performing detailed compositional analysis of snake ven-
oms from genera Porthidium and Cerrophidion. Their distinct
venom toxin compositions also provide clues for rationalizing
the different major signs and symptoms of envenomation by
A. nummifer (predominantly myotoxic) and A. picadoi (mainly
hemorrhagic). Snake venoms are relatively complex mixtures
of toxins belonging however to only 10–13 protein families.
Knowledge on the pathophysiology of envenomation may
suggestthemajortypeoftoxinsinvolved,butactivity-composition
correlations remain speculative until a detailed proteomic
characterization of the venom is carried out. In this sense, the
occurrence of a three-finger toxin in A. nummifer venom was
completely unexpected. The identification of the panel of toxins
putatively involved in the pathophysioloy of snakebites paves
the way for a detailed structural characterization of these
venom proteins, which may find applications in the production
and improvement of antivenoms.
Acknowledgment. This study has been financed by
grant BFU2004-01432/BMC from the Ministerio de Educación
y Ciencia, Madrid, Spain. Traveling between Valencia and San
José was supported by Acciones Integradas Universidad de
Costa Rica-CSIC 2006CR0010.
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